Compositions comprising bacterial strains

ABSTRACT

The invention provides compositions comprising one or more bacterial strains for treating preventing diarrhea and/or constipation.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 4, 2017, isnamed P068687WO_Sequence_Listing.txt and is 12,288 bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 1500 different phylotypesdominated in abundance levels by two major bacterial divisions (phyla),the Bacteroidetes and the Firmicutes [2-3]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host and additional health benefits. Similarly, the immunologicalimportance of the gut microbiota is well-recognized and is exemplifiedin germfree animals which have an impaired immune system that isfunctionally reconstituted following the introduction of commensalbacteria [4-6].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa and Clostridium clusterXI (F. prausnitzii) bacteria are reduced in IBD patients whilst numbersof E. coli are increased, suggesting a shift in the balance of symbiontsand pathobionts within the gut [7-11]. In recognition of the potentialpositive effect that certain bacterial strains may have on the animalgut, various strains have been proposed for use in the treatment ofvarious diseases (see, for example, [12-15]). A number of strains,including mostly Lactobacillus and Bifidobacterium strains, have beenproposed for use in treating various bowel disorders (see [16] for areview). Strains of the genus Blautia have also been proposed for use inmodulating the microbial balance of the digestive ecosystem in IBSpatients (WO 01/85187). However, the relationship between differentbacterial strains and different diseases, and the precise effects ofparticular bacterial strains on the gut and at a systemic level and onany particular types of diseases, are poorly characterised.

There is a requirement for the potential effects of gut bacteria to becharacterised so that new therapies using gut bacteria can be developed.

US 2010/0247489 describes the use of mineral nutrients to treatdigestive disorders. US′789 proposes optionally also using numerousdifferent genera of bacteria to prevent a wide variety of abdominalsymptoms but does not link any bacteria to any diseases or symptoms.

WO 2016/086206 suggests that bacteria in the order Clostridiales can beused to treat or prevent various conditions but does not link anybacteria to any diseases or symptoms.

WO 2012/142605 proposes that it may be possible to treat a number ofdifferent diseases with a combination of microorganisms. WO′605 suggestsa large number of possible bacterial species that could be employed, butthere is no teaching in WO′605 as to how any of the proposed bacterialspecies could be used to treat any of the diseases proposed.

WO 02/07741 suggests using various bacteria from the class Clostridia totreat gastrointestinal disorders but does not link any bacteria to anydiseases or symptoms. WO 2016/086209 suggests using a large number ofpossible bacteria in the order Clostridiales to decrease the secretionof pro-inflammatory cytokines and/or increase the secretion ofanti-inflammatory cytokines, but there is no teaching as to how any ofthe proposed bacterial species could be used to treat any of thediseases proposed.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating and preventingdiarrhea and/or constipation. In particular, the inventors haveidentified that bacterial strains from the genus Blautia can beeffective for reducing diarrhea and/or constipation. As described in theexamples, oral administration of compositions comprising Blautiahydrogenotrophica may reduce diarrhea and/or constipation in patientshaving irritable bowel syndrome (IBS). Therefore, in a first embodiment,the invention provides a composition comprising a bacterial strain ofthe genus Blautia, for use in a method of treating or preventingdiarrhea and/or constipation.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in a methodof treating or preventing diarrhea and/or constipation in a subjectdiagnosed with Crohn's disease, ulcerative colitis, or, more preferably,IBS. In some embodiments, the subject diagnosed with IBS has IBS withboth diarrhea and constipation. In some embodiments, the subjectdiagnosed with IBS has IBS with diarrhea but without constipation orwith only a small amount of constipation. In some embodiments, thesubject diagnosed with IBS has IBS with constipation but withoutdiarrhea or with only a small amount of diarrhea.

In preferred embodiments of the invention, the bacterial strain in thecomposition is of Blautia hydrogenotrophica. Closely related strains mayalso be used, such as bacterial strains that have a 16s rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical tothe 16s rRNA sequence of a bacterial strain of Blautiahydrogenotrophica. Preferably, the bacterial strain has a 16s rRNAsequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to SEQ ID NO:5. Most preferably, the bacterial strain in thecomposition is the Blautia hydrogenotrophica strain deposited underaccession number DSM 10507/14294.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia stercoris. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia stercoris. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 3. Preferably, thesequence identity is to SEQ ID NO:3. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:3.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia wexlerae. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia wexlerae. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or 4. Preferably, thesequence identity is to SEQ ID NO:4. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:4.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for treating diarrhea and/or constipation. Also, oraladministration is convenient for patients and practitioners and allowsdelivery to and/or partial or total colonisation of the intestine.

In certain embodiments, the composition of the invention comprises oneor more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises abacterial strain that has been lyophilised. Lyophilisation is aneffective and convenient technique for preparing stable compositionsthat allow delivery of bacteria, and is shown to provide effectivecompositions in the examples.

In certain embodiments, the invention provides a food product comprisingthe composition as described above.

In certain embodiments, the invention provides a vaccine compositioncomprising the composition as described above.

Additionally, the invention provides a method of treating or preventingdiarrhea and/or constipation, comprising administering a compositioncomprising a bacterial strain of the genus Blautia.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in a methodof treating or preventing a disease associated with Enterobacteriaceaeinfection, such as E. coli infection. In certain embodiments, thecompositions of the invention are for use in treating or preventingdiarrhea, gastroenteritis, urinary tract infection or neonatalmeningitis.

In further preferred embodiments, the compositions of the invention arefor use in reducing the level of Enterobacteriaceae in thegastrointestinal tract, preferably the level of E. coli, in thetreatment or prevention of IBS, Crohn's disease, ulcerative colitis,functional dyspepsia, diarrhea, gastroenteritis, urinary tract infectionor neonatal meningitis.

In particularly preferred embodiments, the compositions of the inventionare for use a method of treating or preventing diarrhea associated withEnterobacteriaceae infection, such as E. coli infection, or are for usein reducing the level of Enterobacteriaceae in the gastrointestinaltract, preferably the level of E. coli, in the treatment or preventionof diarrhea.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Changes in patient symptoms during dosing period (days 1-16) ofPhase I clinical trial.

FIG. 2: Changes in patient symptoms during washout period of Phase Iclinical trial.

FIG. 3a : Hydrogen breath test Cmax results for day 1, day 2, day 15 andday 16 for IBS patients treated with Blautix (FIG. 3a ). FIG. 3b :Hydrogen breath test Cmax results for day 1, day 2, day 15 and day 16for and IBS patients treated with placebo (FIG. 3b ). FIG. 3c is a graphcomparing the percentage of Blautix treated patients with a reduction inhydrogen between the mean of days 15 and 16 and the mean of days 1 and 2to the percentage of placebo treated patients with a reduction inhydrogen between these time points.

FIG. 4a : Hydrogen uncorrected and hydrogen corrected breath test paireddata for day 1 and day 15 for Blautix treated IBS patients; FIG. 4b :graph comparing mean hydrogen uncorrected breath test results for day 1and day 15 for the Blautix treatment group; FIG. 4c : graph comparingmean hydrogen corrected breath test results for day 1 and day 15 for theBlautix treatment group.

FIG. 5a : Hydrogen uncorrected and hydrogen corrected breath test paireddata for day 1 and day 15 for placebo treated IBS patients; FIG. 5b :graph comparing mean hydrogen uncorrected breath test results for day 1and day 15 for the placebo group; FIG. 5c : graph comparing meanhydrogen corrected breath test results for day 1 and day 15 for theplacebo group.

FIG. 6a : Graphs comparing the mean hydrogen breath test results fromday 1 and day 15 for the Bautix treatment group (Verum) and placebogroup for uncorrected hydrogen. FIG. 6b : Graphs comparing the meanhydrogen breath test results from day 1 and day 15 for the Bautixtreatment group (Verum) and placebo group for corrected hydrogen (FIG.6a : uncorrected hydrogen; FIG. 6b : corrected hydrogen).

DISCLOSURE OF THE INVENTION

Bacterial Strains

The compositions of the invention comprise a bacterial strain of thegenus Blautia. The examples demonstrate that bacteria of this genus areuseful for treating or preventing diarrhea and/or constipation. Thepreferred bacterial strains are of the species Blautiahydrogenotrophica, Blautia stercoris and Blautia wexlerae. Otherpreferred bacterial strains for use in the invention are Blautiaproducta, Blautia coccoides and Blautia hansenii.

Examples of Blautia strains for use in the invention include Blautiahydrogenotrophica, B. stercoris, B. faecis, B. coccoides, B. glucerasea,B. hansenii, B. luti, B. producta, B. schinkii and B. wexlerae. TheBlautia species are Gram-reaction-positive, non-motile bacteria that maybe either coccoid or oval and all are obligate anaerobes that produceacetic acid as the major end product of glucose fermentation [17].Blautia may be isolated from the human gut, although B. producta wasisolated from a septicaemia sample.

Blautia hydrogenotrophica (previously known as Ruminococcushydrogenotrophicus) has been isolated from the guts of mammals, isstrictly anaerobic, and metabolises H₂/CO₂ to acetate, which may beimportant for human nutrition and health. The type strain of Blautiahydrogenotrophica is S5a33=DSM 10507=JCM 14656. The GenBank accessionnumber for the 16S rRNA gene sequence of Blautia hydrogenotrophicastrain S5a36 is X95624.1 (disclosed herein as SEQ ID NO:5). Thisexemplary Blautia hydrogenotrophica strain is described in [17] and[18]. The S5a33 strain and the S5a36 strain correspond to two subclonesof a strain isolated from a faecal sample of a healthy subject. Theyshow identical morphology, physiology and metabolism and have identical16S rRNA sequences. Thus, in some embodiments, the Blautiahydrogenotrophica for use in the invention has the 16S rRNA sequence ofSEQ ID NO:5.

The Blautia hydrogenotrophica bacterium deposited under accession numberDSM 10507 and also under accession number DSM 14294 was tested in theExamples and is also referred to herein as strain BH. Strain BH wasdeposited with the Deutsche Sammlung von Mikroorganismen [GermanMicroorganism Collection] (Mascheroder Weg 1b, 38124 Braunschweig,Germany) in January 1996 as “Ruminococcus hydrogenotrophicus” underaccession number DSM 10507 and also under accession number DSM 14294 as“S5a33” on 10 May 2001. The depositor was INRA Laboratoire deMicrobiologie CR de Clermont-Ferrand/Theix 63122 Saint GenesChampanelle, France. Ownership of the deposits has passed to 4D PharmaPlc by way of assignment. The DSM 14294 deposit was made under the termsof the Budapest Treaty. Maintenance of a viable culture is assured for30 years from the date of deposit. All restrictions on the availabilityto the public of the microorganisms deposited as DSM 14294 and DSM 10507will be irrevocably removed upon the granting of a patent for thisapplication.

The GenBank accession number for the 16S rRNA gene sequence of Blautiastercoris strain GAM6-1^(T) is HM626177 (disclosed herein as SEQ IDNO:1). An exemplary Blautia stercoris strain is described in [19]. Thetype strain of Blautia wexlerae is WAL 14507=ATCC BAA-1564=DSM 19850[17]. The GenBank accession number for the 16S rRNA gene sequence ofBlautia wexlerae strain WAL 14507 T is EF036467 (disclosed herein as SEQID NO:2). This exemplary Blautia wexlerae strain is described in [17].

A preferred Blautia stercoris strain is the strain deposited underaccession number NCIMB 42381, which is also referred to herein as strain830. A 16S rRNA sequence for the 830 strain is provided in SEQ ID NO:3.Strain 830 was deposited with the international depositary authorityNCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GTBiologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS,Scotland) on 12 Mar. 2015 as “Blautia stercoris 830” and was assignedaccession number NCIMB 42381. GT Biologics Ltd. subsequently changed itsname to 4D Pharma Research Limited. The NCIMB 42381 deposit was madeunder the terms of the Budapest Treaty. Maintenance of a viable cultureis assured for 30 years from the date of deposit. All restrictions onthe availability to the public of the deposited microorganism will beirrevocably removed upon the granting of a patent for this application.

A preferred Blautia wexlerae strain is the strain deposited underaccession number NCIMB 42486, which is also referred to herein as strainMRX008. A 16S rRNA sequence for the MRX008 strain is provided in SEQ IDNO:4. Strain MRX008 was deposited with the international depositaryauthority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland)by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen,AB25 2ZS, Scotland) on 16 Nov. 2015 as “Blaqutia/Ruminococcus MRx0008”and was assigned accession number NCIMB 424. The NCIMB 42486 deposit wasmade under the terms of the Budapest Treaty. Maintenance of a viableculture is assured for 30 years from the date of deposit. Allrestrictions on the availability to the public of the depositedmicroorganism will be irrevocably removed upon the granting of a patentfor this application.

Bacterial strains closely related to the strain tested in the examplesare also expected to be effective for treating or preventing diarrheaand/or constipation. In certain embodiments, the bacterial strain foruse in the invention has a 16s rRNA sequence that is at least 95%, 96%,97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of abacterial strain of Blautia hydrogenotrophica. Preferably, the bacterialstrain for use in the invention has a 16s rRNA sequence that is at least95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5.

In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to the 16s rRNA sequence of a bacterial strain ofBlautia stercoris. Preferably, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to SEQ ID NO:1 or SEQ ID NO:3. Preferably,the sequence identity is to SEQ ID NO:3. Preferably, the bacterialstrain for use in the invention has the 16s rRNA sequence represented bySEQ ID NO:3. In certain embodiments, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterialstrain of Blautia wexlerae. Preferably, the bacterial strain for use inthe invention has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or SEQ ID NO:4.Preferably, the sequence identity is to SEQ ID NO:4. Preferably, thebacterial strain for use in the invention has the 16s rRNA sequencerepresented by SEQ ID NO:4.

Bacterial strains that are biotypes of the bacterium deposited underaccession number DSM 10507/14294 or biotypes of the bacteria depositedunder accession numbers NCIMB 42381 and NCIMB 42486 are also expected tobe effective for treating or preventing diarrhea and/or constipation. Abiotype is a closely related strain that has the same or very similarphysiological and biochemical characteristics.

Strains that are biotypes of a bacterium deposited under accessionnumber DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and that are suitablefor use in the invention may be identified by sequencing othernucleotide sequences for a bacterium deposited under accession numberDSM 10507/14294, NCIMB 42381 or NCIMB 42486. For example, substantiallythe whole genome may be sequenced and a biotype strain for use in theinvention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%sequence identity across at least 80% of its whole genome (e.g. acrossat least 85%, 90%, 95% or 99%, or across its whole genome). For example,in some embodiments, a biotype strain has at least 98% sequence identityacross at least 98% of its genome or at least 99% sequence identityacross 99% of its genome. Other suitable sequences for use inidentifying biotype strains may include hsp60 or repetitive sequencessuch as BOX, ERIC, (GTG)₅, or REP or [20]. Biotype strains may havesequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenceidentity to the corresponding sequence of a bacterium deposited underaccession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486. In someembodiments, a biotype strain has a sequence with at least 97%, 98%,99%, 99.5% or 99.9% sequence identity to the corresponding sequence ofthe Blautia hydrogenotrophica strain deposited as DSM 10507/14294 andcomprises a 16S rRNA sequence that is at least 99% identical (e.g. atleast 99.5% or at least 99.9% identical) to SEQ ID NO:5. In someembodiments, a biotype strain has a sequence with at least 97%, 98%,99%, 99.5% or 99.9% sequence identity to the corresponding sequence ofthe Blautia hydrogenotrophica strain deposited as DSM 10507/14294 andhas the 16S rRNA sequence of SEQ ID NO:5.

Alternatively, strains that are biotypes of a bacterium deposited underaccession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and thatare suitable for use in the invention may be identified by using theaccession number DSM 10507/14294 deposit, the accession number NCIMB42381 deposit, or the accession number NCIMB 42486 deposit, andrestriction fragment analysis and/or PCR analysis, for example by usingfluorescent amplified fragment length polymorphism (FAFLP) andrepetitive DNA element (rep)-PCR fingerprinting, or protein profiling,or partial 16S or 23s rDNA sequencing. In preferred embodiments, suchtechniques may be used to identify other Blautia hydrogenotrophica,Blautia stercoris or Blautia wexlerae strains.

In certain embodiments, strains that are biotypes of a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486 and that are suitable for use in the invention are strains thatprovide the same pattern as a bacterium deposited under accession numberDSM 10507/14294, NCIMB 42381 or NCIMB 42486 when analysed by amplifiedribosomal DNA restriction analysis (ARDRA), for example when usingSau3AI restriction enzyme (for exemplary methods and guidance see, forexample, p11). Alternatively, biotype strains are identified as strainsthat have the same carbohydrate fermentation patterns as a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486.

Other Blautia strains that are useful in the compositions and methods ofthe invention, such as biotypes of a bacterium deposited under accessionnumber DSM 10507/14294, NCIMB 42381 or NCIMB 42486, may be identifiedusing any appropriate method or strategy, including the assays describedin the examples. For instance, strains for use in the invention may beidentified by culturing bacteria and administering to rats to test inthe distension assay. In particular, bacterial strains that have similargrowth patterns, metabolic type and/or surface antigens to a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486 may be useful in the invention. A useful strain will havecomparable microbiota modulatory activity to the DSM 10507/14294, NCIMB42381 or NCIMB 42486 strain. In particular, a biotype strain will elicitcomparable effects on diarrhea and/or constipation to the effects shownin the Examples, which may be identified by using the culturing andadministration protocols described in the Examples.

A particularly preferred strain of the invention is the Blautiahydrogenotrophica strain deposited under accession number DSM10507/14294. This is the exemplary BH strain tested in the examples andshown to be effective for treating disease. Therefore, the inventionprovides a cell, such as an isolated cell, of the Blautiahydrogenotrophica strain deposited under accession number DSM10507/14294, or a derivative thereof, for use in therapy, in particularfor the diseases described herein.

A derivative of the strain deposited under accession number DSM10507/14294, NCIMB 42381 or NCIMB 42486 may be a daughter strain(progeny) or a strain cultured (subcloned) from the original. Aderivative of a strain of the invention may be modified, for example atthe genetic level, without ablating the biological activity. Inparticular, a derivative strain of the invention is therapeuticallyactive. A derivative strain will have comparable microbiota modulatoryactivity to the original DSM 10507/14294, NCIMB 42381 or NCIMB 42486strain. In particular, a derivative strain will elicit comparableeffects on diarrhea and/or constipation to the effects shown in theExamples, which may be identified by using the culturing andadministration protocols described in the Examples. A derivative of theDSM 10507/14294 strain will generally be a biotype of the DSM10507/14294 strain. A derivative of the NCIMB 42381 strain willgenerally be a biotype of the NCIMB 42381 strain. A derivative of theNCIMB 42486 strain will generally be a biotype of the NCIMB 42486strain.

References to cells of the Blautia hydrogenotrophica strain depositedunder accession number DSM 10507/14294 encompass any cells that have thesame safety and therapeutic efficacy characteristics as the strainsdeposited under accession number DSM 10507/14294, and such cells areencompassed by the invention. References to cells of the Blautiastercoris strain deposited under accession number NCIMB 42381 encompassany cells that have the same safety and therapeutic efficacycharacteristics as the strains deposited under accession number NCIMB42381, and such cells are encompassed by the invention. References tocells of the Blautia wexlerae strain deposited under accession numberNCIMB 42486 encompass any cells that have the same safety andtherapeutic efficacy characteristics as the strains deposited underaccession number NCIMB 42486, and such cells are encompassed by theinvention.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

Therapeutic Uses

In preferred embodiments, the compositions of the invention are for usein treating diarrhea and/or constipation. In some embodiments thecomposition is for use in treating a patient having both diarrhea andconstipation. In some embodiments the composition is for use in treatinga patient having diarrhea without constipation. In some embodiments thecomposition is for use in treating a patient having constipation withoutdiarrhea. Although patients often exhibit either diarrhea orconstipation, a single patient may exhibit both conditions at differenttimes. Patients having IBS and/or inflammatory diseases of the intestineoften exhibit diarrhea and/or constipation. Accordingly, the inventionprovides the composition of the invention for use in treating a patientdiagnosed with or suspected of having IBS or an inflammatory disease ofthe intestine. For example, IBS patients may experience alternatingcycles of diarrhea and constipation or an IBS patient may becharacterised as an IBS patient with diarrhea or an IBS patient withconstipation. In some embodiments, a composition of the invention may beused to treat a patient having IBS, a patient having IBS with diarrheaor a patient having IBS with constipation. For example, in someembodiments, the subject diagnosed with IBS has IBS with both diarrheaand constipation. In some embodiments, the subject diagnosed with IBShas IBS with diarrhea but without constipation or with only a smallamount of constipation. In some embodiments, the subject diagnosed withIBS has IBS with constipation but without diarrhea or with only a smallamount of diarrhea. In some embodiments, the inflammatory disease is ofthe small intestine, colon or rectum. Diarrhea is a symptom of Crohn'sdisease and ulcerative colitis. Accordingly, a composition of theinvention may be used to treat a patient having Crohn's disease orulcerative colitis.

As demonstrated in the examples, bacterial compositions of the inventionmay be effective for reducing diarrhea and/or constipation.

In preferred embodiments, the compositions of the invention are for usein treating or preventing diarrhea and/or constipation associated withCrohn's disease, ulcerative colitis or, more preferably, IBS. Inpreferred embodiments, the compositions of the invention are for use intreating or preventing diarrhea and/or constipation in a subjectdiagnosed with Crohn's disease, ulcerative colitis or, more preferably,IBS. In preferred embodiments the compositions of the invention are foruse in treating or preventing diarrhea and/or constipation in thetreatment of Crohn's disease, ulcerative colitis or, more preferably,IBS. In some embodiments, the patient has abdominal pain and/or bloatingin addition to diarrhea and/or constipation. In such embodiments, thepatient may have any combination of two, three or all four of theseconditions. For example, the patient may have diarrhea, constipation,abdominal pain and bloating; diarrhea, constipation and abdominal pain;diarrhea, constipation and bloating; diarrhea, abdominal pain and/orbloating without constipation; constipation, abdominal pain and/orbloating without diarrhea.

In certain embodiments, the composition may be in the form of abacterial culture. In some embodiments, the composition may preferablybe a lyophilisate.

In some embodiments, the composition of the invention is for use intreating diarrhea and/or constipation in a subject having an increasedlevel of hydrogen in their breath relative to a healthy subject. In someembodiments, the composition of the invention is for use in reducing thehydrogen level in the breath of a subject having diarrhea and/orconstipation. The subject is preferably a subject diagnosed as havingIBS and/or an inflammatory disease of the intestine. In someembodiments, the inflammatory disease is of the small intestine, colonor rectum. In some embodiments, the subject has been diagnosed withCrohn's disease, ulcerative colitis or, more preferably, IBS, forexample one of the types of IBS described herein. Without being bound bytheory, it is speculated that the increased hydrogen level arises fromthe mechanisms underlying the inflammation in the intestine. Theexamples show that treatment with a composition of the invention reducesthe level of hydrogen detected in hydrogen breath tests. Accordingly,the hydrogen levels are preferably assessed using a hydrogen breathtest. The hydrogen breath test is well known in the art and so theskilled person will know how to conduct such a test. In someembodiments, the patient is administered lactulose as the substrate forthe test.

The hydrogen breath test is also a useful tool for monitoring theeffectiveness or likely effectiveness of treatment or prevention using acomposition of an invention. For example, a reduction in the level ofhydrogen detected in a subject's breath following treatment orprevention by a composition of the invention may indicate that thetreatment is having a therapeutic or preventative effect. Accordingly,in some embodiments the methods and uses of the invention furthercomprise monitoring the hydrogen level in a subject's breath duringand/or following treatment or prevention with a composition of theinvention and thereby assessing the effectiveness or likelyeffectiveness of treatment or prevention. For example, hydrogen levelsmay be monitored at one or more (e.g. 1, 2, 3, 4 or more than 4) times,for example, including before treatment, at the start of treatment,during treatment, at the end of treatment and/or following treatment, asdesired. In some embodiments, the level of hydrogen in the subject'sbreath at the end and/or following the dosing period (during which thecomposition is administered to the subject) is compared to the level atthe start and/or before the dosing period and a reduction in the levelindicates the effectiveness or likely effectiveness of the treatment orprevention. For example, in embodiments in which the dosing period is 16days, it may be desirable to take measurements at day 1 and day 16, orfor example at day 1, day 2, day 15 and day 16. In some embodiments,multiple measurements are taken and the mean of those measurementsobtained (for example, the mean of day 1 and day 2 and the mean of day15 and day 16). In some embodiments, a reduction in at least 40 ppm inthe hydrogen level Cmax indicates that the treatment or prevention iseffective or likely to be effective. In some embodiments, the hydrogenlevel in the subject's breath is measured only once, for example, at theend of or following treatment, and the finding that the level is at orclose to a predetermined level is indicative that the treatment orprevention is likely to have been effective. The hydrogen breath test isa standard assay and so predetermined levels are known in the art.

In certain embodiments, the compositions of the invention are for use inpreventing diarrhea and/or constipation in a subject that is receivingor has received or is about to receive (for example, later the same day,the next day, or within 2, 3, 4, 5, 6, 7, 10, 14 or 21 days) antibiotictreatment or that is suffering from or has suffered from bacterialgastroenteritis. Antibiotic treatment and bacterial gastroenteritis areassociated with changes in the gut microbiota that may precede diarrheaand/or constipation and that may be prevented by the compositions of theinvention. The compositions of the invention may be administeredsimultaneously, separately or sequentially with an antibiotic treatment.For example, the compositions of the invention may be administeredconcurrently with an antibiotic treatment.

In preferred embodiments, treatment with compositions of the inventionresults in a reduction in diarrhea and/or constipation.

Treatment or prevention of diarrhea and/or constipation may refer to,for example, an alleviation of the severity of symptoms or a reductionin the frequency of exacerbations or the range of triggers that are aproblem for the patient.

In certain embodiments, the compositions of the invention are for use intreating or preventing a disease associated with Enterobacteriaceaeinfection, such as E. coli infection. In certain embodiments, thecompositions of the invention are for use in treating or preventingdiarrhea, gastroenteritis, urinary tract infection or neonatalmeningitis. In such embodiments the Enterobacteriaceae may be apathogenic strain.

In certain embodiments, the compositions of the invention are for use intreating or preventing a disease associated with increased levels ofEnterobacteriaceae, such as E. coli. In certain embodiments, thecompositions of the invention are for use in treating or preventingdiarrhea, gastroenteritis, urinary tract infection or neonatalmeningitis. In such embodiments, the Enterobacteriaceae may be acommensal or non-pathogenic strain.

In preferred embodiments, the compositions of the invention are for usein reducing the level of Enterobacteriaceae in the gastrointestinaltract, preferably the level of E. coli, in the treatment or preventionof a disease associated with increased levels of Enterobacteriaceae,such as IBS, Crohn's disease, ulcerative colitis, functional dyspepsia,diarrhea, gastroenteritis, urinary tract infection or neonatalmeningitis. Enterobacteriaceae and in particular E. coli are known to bepotential triggers for, or known to exacerbate, Crohn's disease andulcerative colitis [22-24], so the effect shown in the examples for thecompositions of the invention may be beneficial in the treatment ofthese conditions.

In certain embodiments, the compositions of the invention are for use intreating or preventing IBS, Crohn's disease, ulcerative colitis,functional dyspepsia, diarrhea, gastroenteritis, urinary tract infectionor neonatal meningitis by reducing the level of Enterobacteriaceae inthe gastrointestinal tract.

Modes of administration Preferably, the compositions of the inventionare to be administered to the gastrointestinal tract in order to enabledelivery to and/or partial or total colonisation of the intestine withthe bacterial strain of the invention. Generally, the compositions ofthe invention are administered orally, but they may be administeredrectally, intranasally, or via buccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily. The examples demonstrate that administration provides successfulcolonisation and clinical benefits in treatment of diarrhea and/orconstipation.

In certain embodiments, the compositions of the invention areadministered regularly, such as daily, every two days, or weekly, for anextended period of time, such as for at least one week, two weeks, onemonth, two months, six months, or one year. The examples demonstratethat BH administration may not result in permanent colonisation of theintestines, so regular administration for extended periods of time mayprovide greater therapeutic benefits.

In some embodiments the compositions of the invention are administeredfor 7 days, 14 days, 16 days, 21 days or 28 days or no more than 7 days,14 days, 16 days, 21 days or 28 days. For example, in some embodimentsthe compositions of the invention are administered for 16 days.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to prevent diarrhea and/or constipation developing in her childin utero and/or after it is born.

The compositions of the invention may be administered to a patient thathas been diagnosed with diarrhea and/or constipation or a disease orcondition associated with diarrhea and/or constipation, or that has beenidentified as being at risk of diarrhea and/or constipation. Thecompositions may also be administered as a prophylactic measure toprevent the development of diarrhea and/or constipation in a healthypatient.

The compositions of the invention may be administered to a patient thathas been identified as having an abnormal gut microbiota. For example,the patient may have reduced or absent colonisation by Blautia, and inparticular Blautia hydrogenotrophica, Blautia stercoris or Blautiawexlerae.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

In some embodiments, the subject to whom the composition is to beadministered is an adult human.

In some embodiments, the subject to whom the composition is to beadministered is an infant human.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilisedbacteria. Lyophilisation of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [25-27]Lyophilisate compositions may be particularly effective. In preferredembodiments, the compositions of the invention comprises lyophilisedbacteria and is for the treatment of diarrhea and/or constipationassociated with IBS.

Alternatively, the composition of the invention may comprise a live,active bacterial culture. The examples demonstrate that cultures of thebacteria of the invention are therapeutically effective.

In some embodiments, the bacterial strain in the composition of theinvention has not been inactivated, for example, has not beenheat-inactivated. In some embodiments, the bacterial strain in thecomposition of the invention has not been killed, for example, has notbeen heat-killed. In some embodiments, the bacterial strain in thecomposition of the invention has not been attenuated, for example, hasnot been heat-attenuated. For example, in some embodiments, thebacterial strain in the composition of the invention has not beenkilled, inactivated and/or attenuated. For example, in some embodiments,the bacterial strain in the composition of the invention is live. Forexample, in some embodiments, the bacterial strain in the composition ofthe invention is viable. For example, in some embodiments, the bacterialstrain in the composition of the invention is capable of partially ortotally colonising the intestine. For example, in some embodiments, thebacterial strain in the composition of the invention is viable andcapable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of livebacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [28-29].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becauseBlautia are anaerobes. Other ingredients (such as vitamin C, forexample), may be included as oxygen scavengers and prebiotic substratesto improve the delivery and/or partial or total colonisation andsurvival in vivo. Alternatively, the probiotic composition of theinvention may be administered orally as a food or nutritional product,such as milk or whey based fermented dairy product, or as apharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a patient. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU; in another example from about 1×10⁷ toabout 1×10¹¹ CFU; in another example from about 1×10⁸ to about 1×10¹⁰CFU; in another example from about 1×10⁸ to about 1×10¹¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10⁹ cellsper day, such as at least 10¹⁰, at least 10¹¹, or at least 10¹² cellsper day.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [30]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [31]. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid, cysteine and estersof p-hydroxybenzoic acid, for example, in some embodiments thepreservative is selected from sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused. A further example of a suitable carrier is saccharose. A furtherexample of a preservative is cysteine.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated as a milk-based product. Theterm “milk-based product” means any liquid or semi-solid milk- orwhey-based product having a varying fat content. The milk-based productcan be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, wholemilk, milk recombined from powdered milk and whey without anyprocessing, or a processed product, such as yoghurt, curdled milk, curd,sour milk, sour whole milk, butter milk and other sour milk products.Another important group includes milk beverages, such as whey beverages,fermented milks, condensed milks, infant or baby milks; flavoured milks,ice cream; milk-containing food such as sweets.

In some embodiments, the compositions of the invention comprise one ormore bacterial strains of the genus Blautia and do not contain bacteriafrom any other genus, or which comprise only de minimis or biologicallyirrelevant amounts of bacteria from another genus.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism. In some embodiments, such compositionsmay be a lyophilisate that is substantially free from other species oforganism.

In certain embodiments, the compositions of the invention comprise oneor more bacterial strains of the genus Blautia, for example, a Blautiahydrogenotrophica, and do not contain any other bacterial genus, orwhich comprise only de minimis or biologically irrelevant amounts ofbacteria from another genus. In certain embodiments, the compositions ofthe invention comprise a single species of Blautia, for example, aBlautia hydrogenotrophica, and do not contain any other bacterialspecies, or which comprise only de minimis or biologically irrelevantamounts of bacteria from another species. In certain embodiments, thecompositions of the invention comprise a single strain of Blautia, forexample, of Blautia hydrogenotrophica, and do not contain any otherbacterial strains or species, or which comprise only de minimis orbiologically irrelevant amounts of bacteria from another strain orspecies.

In some embodiments, the compositions of the invention comprise morethan one bacterial strain or species. For example, in some embodiments,the compositions of the invention comprise more than one strain fromwithin the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not containbacteria from any other species. In some embodiments, the compositionsof the invention comprise less than 50 strains from within the samespecies (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6,5, 4 or 3 strains), and, optionally, do not contain bacteria from anyother species. In some embodiments, the compositions of the inventioncomprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15,16-25, or 31-50 strains from within the same species and, optionally, donot contain bacteria from any other species. In some embodiments, thecompositions of the invention comprise more than one species from withinthe same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not containbacteria from any other genus. In some embodiments, the compositions ofthe invention comprise less than 50 species from within the same genus(e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or3 species), and, optionally, do not contain bacteria from any othergenus.

In some embodiments, the compositions of the invention comprise 1-50,1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2,2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50species from within the same genus and, optionally, do not containbacteria from any other genus. The invention comprises any combinationof the foregoing.

In some embodiments, the composition comprises a microbial consortium.For example, in some embodiments, the composition comprises the Blautiabacterial strain as part of a microbial consortium. For example, in someembodiments, the Blautia bacterial strain is present in combination withone or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterialstrains from other genera with which it can live symbiotically in vivoin the intestine. For example, in some embodiments, the compositioncomprises a bacterial strain of Blautia hydrogenotrophica in combinationwith a bacterial strain from a different genus. In some embodiments, themicrobial consortium comprises two or more bacterial strains obtainedfrom a faeces sample of a single organism, e.g. a human. In someembodiments, the microbial consortium is not found together in nature.For example, in some embodiments, the microbial consortium comprisesbacterial strains obtained from faeces samples of at least two differentorganisms. In some embodiments, the two different organisms are from thesame species, e.g. two different humans. In some embodiments, the twodifferent organisms are an infant human and an adult human. In someembodiments, the two different organisms are a human and a non-humanmammal.

In some embodiments, the composition of the invention additionallycomprises a bacterial strain that has the same safety and therapeuticefficacy characteristics as the Blautia hydrogenotrophica straindeposited under accession number DSM 10507/14294, but which is not theBlautia hydrogenotrophica strain deposited under accession number DSM10507/14294, or which is not a Blautia hydrogenotrophica or which is nota Blautia.

In some embodiments in which the composition of the invention comprisesmore than one bacterial strain, species or genus, the individualbacterial strains, species or genera may be for separate, simultaneousor sequential administration. For example, the composition may compriseall of the more than one bacterial strain, species or genera, or thebacterial strains, species or genera may be stored separately and beadministered separately, simultaneously or sequentially. In someembodiments, the more than one bacterial strains, species or genera arestored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention isobtained from human adult faeces. In some embodiments in which thecomposition of the invention comprises more than one bacterial strain,all of the bacterial strains are obtained from human adult faeces or ifother bacterial strains are present they are present only in de minimisamounts. The bacteria may have been cultured subsequent to beingobtained from the human adult faeces and being used in a composition ofthe invention.

In some embodiments, the one or more Blautia bacterial strains is/arethe only therapeutically active agent(s) in a composition of theinvention. In some embodiments, the bacterial strain(s) in thecomposition is/are the only therapeutically active agent(s) in acomposition of the invention.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is spray dried. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is live. In certain embodiments, the invention providesthe above pharmaceutical composition, wherein the bacterial strain islyophilised or spray dried and wherein it is viable. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is capable of partially or totally colonising theintestine. In certain embodiments, the invention provides the abovepharmaceutical composition, wherein the bacterial strain is lyophilisedor spray dried and wherein it is viable and capable of partially ortotally colonising the intestine.

In some cases, the lyophilised or spray dried bacterial strain isreconstituted prior to administration. In some cases, the reconstitutionis by use of a diluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to treat a disorder whenadministered to a subject in need thereof; and wherein the disorder isdiarrhea and/or constipation, such as diarrhea and/or constipationassociated with Crohn's disease, ulcerative colitis or, more preferably,IBS.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, there is provided the pharmaceutical compositionof the invention, wherein the composition does not comprise anyminerals, or more specifically, does not comprise any metals with anatomic number greater than 33, for example, wherein the composition doesnot comprise any minerals from the group consisting of selenium,molybdenum, tungsten, selenium compounds, molybdenum compounds andtungsten compounds.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4.0 or about 25.0 and the container is placed in anatmosphere having 50% relative humidity, at least 80% of the bacterialstrain as measured in colony forming units, remains after a period of atleast about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years,2.5 years or 3 years.

In some embodiments, the composition of the invention is provided in asealed container comprising a composition as described herein. In someembodiments, the sealed container is a sachet or bottle. In someembodiments, the composition of the invention is provided in a syringecomprising a composition as described herein.

The composition of the present invention may, in some embodiments, beprovided as a pharmaceutical formulation. For example, the compositionmay be provided as a tablet or capsule. In some embodiments, the capsuleis a gelatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are administeredorally. Oral administration may involve swallowing, so that the compoundenters the gastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth.

Pharmaceutical formulations suitable for oral administration includesolid plugs, solid microparticulates, semi-solid and liquid (includingmultiple phases or dispersed systems) such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids (e.g. aqueoussolutions), emulsions or powders; lozenges (including liquid-filled);chews; gels; fast dispersing dosage forms; films; ovules; sprays; andbuccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an entericformulation, i.e. a gastro-resistant formulation (for example, resistantto gastric pH) that is suitable for delivery of the composition of theinvention to the intestine by oral administration. Enteric formulationsmay be particularly useful when the bacteria or another component of thecomposition is acid-sensitive, e.g. prone to degradation under gastricconditions.

In some embodiments, the enteric formulation comprises an entericcoating. In some embodiments, the formulation is an enteric-coateddosage form. For example, the formulation may be an enteric-coatedtablet or an enteric-coated capsule, or the like. The enteric coatingmay be a conventional enteric coating, for example, a conventionalcoating for a tablet, capsule, or the like for oral delivery. Theformulation may comprise a film coating, for example, a thin film layerof an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric,for example, gastro-resistant without the need for an enteric coating.Thus, in some embodiments, the formulation is an enteric formulationthat does not comprise an enteric coating. In some embodiments, theformulation is a capsule made from a thermogelling material. In someembodiments, the thermogelling material is a cellulosic material, suchas methylcellulose, hydroxymethylcellulose orhydroxypropylmethylcellulose (HPMC). In some embodiments, the capsulecomprises a shell that does not contain any film forming polymer. Insome embodiments, the capsule comprises a shell and the shell compriseshydroxypropylmethylcellulose and does not comprise any film formingpolymer (e.g. see [32]). In some embodiments, the formulation is anintrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsulesare capsules which may, owing to additions of softeners, such as, forexample, glycerol, sorbitol, maltitol and polyethylene glycols, presentin the capsule shell, have a certain elasticity and softness. Softcapsules can be produced, for example, on the basis of gelatine orstarch. Gelatine-based soft capsules are commercially available fromvarious suppliers. Depending on the method of administration, such as,for example, orally or rectally, soft capsules can have various shapes,they can be, for example, round, oval, oblong or torpedo-shaped. Softcapsules can be produced by conventional processes, such as, forexample, by the Scherer process, the Accogel process or the droplet orblowing process.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [33-35].

The solid or liquid medium used for culture may for example be YCFA agaror YCFA medium. YCFA medium may include (per 100 ml, approximatevalues): Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g),cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g),(NH₄)₂SO₄ (0.09 g), MgSO₄.7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin(0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoicacid (3 μg), folic acid (5 μg), and pyridoxamine (15 μg).

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[36-43], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref[44]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref [45].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—Changes in PatientSymptoms During Phase I Clinical Trial

A Phase I clinical trial was conducted in which Blautiahydrogenotrophica (“Blautix”, strain deposited under accession numberDSM 10507 and also under accession number DSM 14294) was administered tohuman patients having irritable bowel syndrome (IBS). Patients wereadministered Blautix during a dosing period (days 1-16) with the washoutperiod being day 19-23. Blautix was found to be both safe and welltolerated. Four symptoms were monitored, of which two were diarrhea andconstipation. The study recorded whether patients experienced animprovement in, no change in or worsening of each of these symptoms.Results from patients administered Blautix were compared with thoseobtained using patients administered a placebo. Symptoms were monitoredat three time points: day 1, day 15/16 and at the end of the study. Theresults are shown in FIGS. 1 and 2.

When the patients' reported symptoms at day 16 were compared to thebaseline from day 1, 82% of 17 IBS patients receiving Blautix reportedan improvement in symptoms (FIG. 1). Improvement of symptoms, two ofwhich are diarrhea and constipation, supports the use of Blautix fortreating or preventing diarrhea and/or constipation.

50% of patients receiving placebo reported an improvement in symptoms(FIG. 1). High placebo response rates are an established phenomenon inIBS clinical studies. Xifaxan was recently approved to treat IBS basedon much smaller improvements over placebo (see:http://www.accessdata.fda.gov/spl/data/5ab6fceb-4d22-4480-81fc-8bc28c16770d/5ab6fceb-4d22-4480-81fc-8bc28c16770d.xml).

A worsening of symptoms at the study completion (day 19-23) compared tosymptoms present upon dosing completion (day 16) is expected based onthe teaching presented here. This worsening of symptoms was seen in thePhase I clinical trial: 41% of IBS patients reported worsening ofsymptoms following cessation of Blautix dosing (FIG. 2). The worseningof symptoms, two of which are diarrhea and constipation, followingcessation of Blautix dosing therefore also supports the use of Blautixin treating or preventing diarrhea and/or constipation.

Example 2—Hydrogen Breath Test Results

Breath hydrogen levels are a biomarker of Blautix activity—MoA involvesmetabolism of endogenous H₂ to produce acetate. Human subjects wereadministered lactulose and hydrogen (H₂) levels (Cmax) were sampled atfour time points: day 1, day 2, day 15 and day 16. Hydrogen uncorrectedresults were converted to hydrogen corrected results.

Some patients were excluded from the analysis. There were three reasonswhy a subject was not included in the hydrogen breath test analysis: 1)They produced a CMAX hydrogen breath test result of <20 on one of thefour sampling days and were therefore deemed to have not responded tothe test; 2) They were methane producers (taken as producing moremethane than hydrogen in the breath test), this affects the hydrogenresponse; and/or 3) there was an aberrant value obtained in the hydrogenbreath test (232 ppm). Subjects 3.12 (Blautix), 3.24 (Blautix), 4.07(Blautix) were excluded as non-responders. Subjects 3.03 (Blautix) and3.08 (Placebo) were excluded as methane producers (4.07, mentioned asexcluded above, was also a methane producer). Subject 4.09 (Placebo) wasexcluded due to an aberrant value.

The results of the corrected hydrogen analysis from the end of thedosing period (day 15/16) were compared to those from the baseline (day½). 10 out of 12 patients (83%) receiving Blautix had a reduction inhydrogen levels over this period (FIGS. 3a and 3c ). In contrast, 3 outof 6 (50%) patients receiving placebo had reduced hydrogen levels (FIGS.3b and 3c ). These percentages are similar to the percentages ofpatients exhibiting an improvement in symptoms following Blautixtreatment or administration of placebo.

FIG. 4 shows the uncorrected and corrected hydrogen results for theBlautix (Verum) treatment group together with a statistical analysis ofthe results. The mean values for both uncorrected and corrected H₂ werefound to differ between day 1 and day 15. After 13.5 days of treatment,a statistically significant (p<0.05) decrease of H₂ in Cmax breath testwas detected after lactulose stimulation. In contrast, for the placebogroup, the mean was found to be equivalent for day 1 and day 15 (p>0.05)(FIG. 5). Thus, the mean for the treatment group (also referred to asthe VERUM group), decreases between day 1 and day 15 whereas the meanfor the placebo group is equivalent between day 1 and day 15 for boththe uncorrected hydrogen results and the corrected hydrogen results(FIGS. 6A and 6B).

Example 3—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Sequences

(Blautia stercoris strain GAM6-1 16S ribosomal RNA gene,partial sequence - HM626177) SEQ ID NO: 1    1tgcaagtcga gcgaagcgct tacgacagaa ccttcggggg aagatgtaag ggactgagcg   61gcggacgggt gagtaacgcg tgggtaacct gcctcataca gggggataac agttggaaac  121ggctgctaat accgcataag cgcacggtat cgcatgatac agtgtgaaaa actccggtgg  181tatgagatgg acccgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg  241atcagtagcc ggcctgagag ggtgaacggc cacattggga ctgagacacg gcccagactc  301ctacgggagg cagcagtggg gaatattgca caatggggga aaccctgatg cagcgacgcc  361gcgtgaagga agaagtatct cggtatgtaa acttctatca gcagggaaga aaatgacggt  421acctgactaa gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca  481agcgttatcc ggatttactg ggtgtaaagg gagcgtagac ggaagagcaa gtctgatgtg  541aaaggctggg gcttaacccc aggactgcat tggaaactgt ttttcttgag tgccggagag  601gtaagcggaa ttcctagtgt agcggtgaaa tgcgtagata ttaggaggaa caccagtggc  661gaaggcggct tactggacgg taactgacgt tgaggctcga aagcgtgggg agcaaacagg  721attagatacc ctggtagtcc acgccgtaaa cgatgaatac taggtgttgg ggagcaaagc  781tcttcggtgc cgcagcaaac gcaataagta ttccacctgg ggagtacgtt cgcaagaatg  841aaactcaaag gaattgacgg ggacccgcac aagcggtgga gcatgtggtt taattcgaag  901caacgcgaag aaccttacca agtcttgaca tcgatctgac cggttcgtaa tggaaccttt  961ccttcgggac agagaagaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1021gggttaagtc ccgcaacgag cgcaacccct atcctcagta gccagcaggt gaagctgggc 1081actctgtgga gactgccagg gataacctgg aggaaggcgg ggacgacgtc aaatcatcat 1141gccccttatg atttgggcta cacacgtgct acaatggcgt aaacaaaggg aagcgagccc 1201gcgaggggga gcaaatccca aaaataacgt cccagttcgg actgcagtct gcaactcgac 1261tgcacgaagc tggaatcgct agtaatcgcg aatcagaatg tcgcggtgaa tacgttcccg 1321ggtcttgtac acaccgcccg tcacaccatg ggagtcagta acgcccgaag tc

(Blautia wexlerae strain WAL 14507 16S ribosomal RNA gene,partial sequence - EF036467) SEQ ID NO: 2    1caagtcgaac gggaattant ttattgaaac ttcggtcgat ttaatttaat tctagtggcg   61gacgggtgag taacgcgtgg gtaacctgcc ttatacaggg ggataacagt cagaaatggc  121tgctaatacc gcataagcgc acagagctgc atggctcagt gtgaaaaact ccggtggtat  181aagatggacc cgcgttggat tagcttgttg gtggggtaac ggcccaccaa ggcgacgatc  241catagccggc ctgagagggt gaacggccac attgggactg agacacggcc cagactccta  301cgggaggcag cagtggggaa tattgcacaa tgggggaaac cctgatgcag cgacgccgcg  361tgaaggaaga agtatctcgg tatgtaaact tctatcagca gggaagatag tgacggtacc  421tgactaagaa gccccggcta actacgtgcc agcagccgcg gtaatacgta gggggcaagc  481gttatccgga tttactgggt gtaaagggag cgtagacggt gtggcaagtc tgatgtgaaa  541ggcatgggct caacctgtgg actgcattgg aaactgtcat acttgagtgc cggaggggta  601agcggaattc ctagtgtagc ggtgaaatgc gtagatatta ggaggaacac cagtggcgaa  661ggcggcttac tggacggtaa ctgacgttga ggctcgaaag cgtggggagc aaacaggatt  721agataccctg gtagtccacg ccgtaaacga tgaataacta ggtgtcgggt ggcaaagcca  781ttcggtgccg tcgcaaacgc agtaagtatt ccacctgggg agtacgttcg caagaatgaa  841actcaaagga attgacgggg acccgcacaa gcggtggagc atgtggttta attcgaagca  901acgcgaagaa ccttaccaag tcttgacatc cgcctgaccg atccttaacc ggatctttcc  961ttcgggacag gcgagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1021gttaagtccc gcaacgagcg caacccctat cctcagtagc cagcatttaa ggtgggcact 1081ctggggagac tgccagggat aacctggagg aaggcgggga tgacgtcaaa tcatcatgcc 1141ccttatgatt tgggctacac acgtgctaca atggcgtaaa caaagggaag cgagattgtg 1201agatggagca aatcccaaaa ataacgtccc agttcggact gtagtctgca acccgactac 1261acgaagctgg aatcgctagt aatcgcggat cagaatgccg cggtgaatac gttcccgggt 1321cttgtacaca ccgcccgtca caccatggga gtcagtaacg cccgaagtca gtgacctaac 1381tgcaaagaag gagctgccga aggcgggacc gatgactggg gtgaagtcgt aacaaggt

(consensus 16S rRNA sequence for Blautia stercoris strain 830)SEQ ID NO: 3 TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTACGACAGAACCTTCGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACAGTTGGAAACGGCTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCATTGGAAACTGTTTTTCTTGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAGCAAAGCTCTTCGGTGCCGCAGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCGATCTGACCGGTTCGTAATGGAACCTTTCCTTCGGGACAGAGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGCCCGCGAGGGGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTAGGGAGGGAGCTGCCGAAGGCGGGATTGATAACTGGGGTGAAGTCTAGGGGGT

(consensus 16S rRNA sequence for Blautia wexlerae strain MRX008)SEQ ID NO: 4 TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTTATACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGCGCACAGAGCTGCATGGCTCAGTGTGAAAAACTCCGGTGGTATAAGATGGACCCGCGTTGGATTAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGATCCATAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAACCTGTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCGGTGCCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCGCCTGACCGATCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGATCGTGAGATGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGTAGTCTGCAACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCTAACTGCAAAGAAGGAGCTGCCGAA

(Blautia hydrogenotrophica strain S5a36 16S ribosomal RNA gene,partial sequence- X95624.1) SEQ ID NO: 5    1gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gaagcgatag agaacggaga   61tttcggttga agttttctat tgactgagtg gcggacgggt gagtaacgcg tgggtaacct  121gccctataca gggggataac agttagaaat gactgctaat accgcataag cgcacagctt  181cgcatgaagc ggtgtgaaaa actgaggtgg tataggatgg acccgcgttg gattagctag  241ttggtgaggt aacggcccac caaggcgacg atccatagcc ggcctgagag ggtgaacggc  301cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg gaatattgca  361caatggggga aaccctgatg cagcgacgcc gcgtgaagga agaagtatct cggtatgtaa  421acttctatca gcagggaaga aagtgacggt acctgactaa gaagccccgg ctaattacgt  481gccagcagcc gcggtaatac gtaaggggca agcgttatcc ggatttactg ggtgtaaagg  541gagcgtagac ggtttggcaa gtctgatgtg aaaggcatgg gctcaacctg tggactgcat  601tggaaactgt cagacttgag tgccggagag gcaagcggaa ttcctagtgt agcggtgaaa  661tgcgtagata ttaggaggaa caccagtggc gaaggcggcc tgctggacgg taactgacgt  721tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa  781cgatgaatac taggtgtcgg gtggcaaagc cattcggtgc cgcagcaaac gcaataagta  841ttcccacctg gggagtacgt tcgcaagaat gaaactcaaa ggaattgacg gggacccgca  901caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aaatcttgac  961atccctctga ccgggaagta atgttccctt ttcttcggaa cagaggagac aggtggtgca 1021tggttgtcg tcagctcgtg tcgtgagatg ttgggttaagt cccgcaacg agcgcaaccct 1081tattcttag tagccagcag gtagagctgg gcactctaggg agactgcca gggataacctg 1141gaggaaggt ggggatgacg tcaaatcatc atgccccttat gatttgggc tacacacgtgc 1201tacaatggc gtaaacaaag ggaagcgaag gggtgacctgg agcaaatct caaaaataacg 1261tctcagttc ggattgtagt ctgcaactcg actacatgaag ctggaatcg ctagtaatcgc 1321gaatcagaa tgtcgcggtg aatacgttcc cgggtcttgta cacaccgcc cgtcacaccat 1381gggagtcag taacgcccga agtcagtgac ccaaccnaaag gagggagct gccgaaggtgg 1441gactgataa ctggggtga

REFERENCES

-   [1] Spor et al. (2011) Nat Rev Microbiol. 9(4):279-90.-   [2] Eckburg et al. (2005) Science. 10; 308(5728):1635-8.-   [3] Tap et al. (2009) Environ Microbiol, 11(10):2574-84-   [4] Macpherson et al. (2001) Microbes Infect. 3(12):1021-35-   [5] Macpherson et al. (2002) Cell Mol Life Sci. 59(12):2088-96.-   [6] Mazmanian et al. (2005) Cell 15; 122(1):107-18.-   [7] Frank et al. (2007) PNAS 104(34):13780-5.-   [8] Scanlan et al. (2006) J Clin Microbiol. 44(11):3980-8.-   [9] Kang et al. (2010) Inflamm Bowel Dis. 16(12):2034-42.

[10] Machiels et al. (2013) Gut. 63(8):1275-83.

-   [11] Lopetuso et al. (2013), Gut Pathogens, 5: 23-   [12] WO 2013/050792-   [13] WO 03/046580-   [14] WO 2013/008039-   [15] WO 2014/167338-   [16] Lee and Lee (2014) World J Gastroenterol. 20(27): 8886-8897.-   [17] Liu et al. (2008) Int J Syst Evol Microbiol 58, 1896-1902.-   [18] Bernalier et al. (1996) Arch. Microbiol. 166 (3), 176-183.-   [19] Park et al. (2012) Int J Syst Evol Microbiol. 62(Pt 4):776-9.-   [20] Masco et al. (2003) Systematic and Applied Microbiology,    26:557-563.-   [21] Sråtková et al. (2011) J. Microbiol. Methods, 87(1): 10-6.-   [22] Darfeuille-Michaud et al. (2004) Gastroenterology    127(2):412-21.-   [23] Strus et al. (2015) Cent Eur J Immunol. 40(4):420-30.-   [24] Petersen et al. (2015) Scand J Gastroenterol.; 50(10):1199-207.-   [25] Miyamoto-Shinohara et al. (2008) J. Gen. Appl. Microbiol., 54,    9-24.-   [26] Cryopreservation and Freeze-Drying Protocols, ed. by Day and    McLellan, Humana Press.-   [27] Leslie et al. (1995) Appl. Environ. Microbiol. 61, 3592-3597.-   [28] Mitropoulou et al. (2013) J Nutr Metab. (2013) 716861.-   [29] Kailasapathy et al. (2002) Curr Issues Intest Microbiol.    3(2):39-48.-   [30] Handbook of Pharmaceutical Excipients, 2nd Edition, (1994),    Edited by A Wade and PJ Weller-   [31] Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.    Gennaro edit. 1985)-   [32] US 2016/0067188-   [33] Handbook of Microbiological Media, Fourth Edition (2010) Ronald    Atlas, CRC Press.-   [34] Maintaining Cultures for Biotechnology and Industry (1996)    Jennie C. Hunter-Cevera, Academic Press-   [35] Strobel (2009) Methods Mol Biol. 581:247-61.-   [36] Gennaro (2000) Remington: The Science and Practice of Pharmacy.    20th edition, ISBN: 0683306472.-   [37] Molecular Biology Techniques: An Intensive Laboratory Course,    (Ream et al., eds., 1998, Academic Press).-   [38] Methods In Enzymology (S. Colowick and N. Kaplan, eds.,    Academic Press, Inc.)-   [39] Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir    and C. C. Blackwell, eds, 1986, Blackwell Scientific Publications)-   [40] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual,    3rd edition (Cold Spring Harbor Laboratory Press).-   [41] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed.,    CRC Press, 1997)-   [42] Ausubel et al. (eds) (2002) Short protocols in molecular    biology, 5th edition (Current Protocols).-   [43] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton &    Graham eds., 1997, Springer Verlag)-   [44] Current Protocols in Molecular Biology (F. M. Ausubel et al.,    eds., 1987) Supplement 30-   [45] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.

The invention claimed is:
 1. A method of treating diarrhea, constipation, or diarrhea and constipation in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising: a bacteria strain of Blautia hydrogenotrophica sufficient to treat the diarrhea, constipation, or diarrhea and constipation in the subject; and a pharmaceutically acceptable excipient, diluent, or carrier, wherein the bacteria strain comprises a 16S rRNA gene sequence of SEQ ID NO: 5, and wherein the administering results in a reduction in the diarrhea, constipation, or diarrhea and constipation in the subject.
 2. The method of claim 1, wherein the bacteria strain is active.
 3. The method of claim 1, wherein the pharmaceutical composition is encapsulated.
 4. The method of claim 1, wherein the pharmaceutical composition is lyophilized.
 5. The method of claim 1, wherein the pharmaceutical composition is formulated for oral delivery.
 6. The method of claim 5, wherein the pharmaceutical composition is enterically coated.
 7. The method of claim 1, wherein the pharmaceutical composition comprises from about 1×10³ to about 1×10¹¹ colony forming units (CFU)/g of the bacteria strain with respect to a total weight of the pharmaceutical composition.
 8. The method of claim 1, wherein the subject has diarrhea and constipation.
 9. The method of claim 1, wherein the pharmaceutical composition comprises an amount sufficient to reduce an amount of hydrogen in the breath of the subject as determined by an administration of lactulose followed by a hydrogen breath analysis.
 10. The method of claim 1, wherein the administering comprises providing one or more doses of 1 g, 3 g, 5 g or 10 g of the pharmaceutical composition.
 11. The method of claim 1, wherein at least 50% of the bacteria strain as measured by an amount of colony forming units (CFU) remains viable after about 1 year of storage when the pharmaceutical composition is stored in a closed container at 25° C. at 95% relative humidity. 